Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay

Summary An IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGMI, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGMI and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.     

Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti-C. muytjensii immunoglobulin G (IgG) and chicken anti-C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti-C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme-linked immunosorbent assay were using rabbit anti-C. muytjensii IgG (1 μg/mL) as a detection antibody and chicken anti-C. muytjensii IgY (10 μg/mL) as a capture antibody. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross-reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 × 10(4) colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 × 10(4) CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.     

Enzyme-linked immunosorbent assay for the determination of p21-activated kinase activity

An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the cross-linked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP.Mg(2+), and detecting peptide phosphorylation using the phospho-epitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC(50) value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.     

抗哇巴因鸡蛋黄IgY与兔抗体IgG在酶联免疫检测中的比较

目的：探讨提高内源性哇巴因（EO）检测方法的准确性及特异性,为哇巴因的研究打下基础。方法：分别制备出鸡蛋黄抗哇巴因多克隆抗体及兔抗体。然后采用酶联免疫吸附法（ELISA）比较两种抗体检测内源性哇巴因的准确性及特异性。结果：采用IgY检测EO的平均批内变异系数为2．03％,批间变异系数为2．34％。兔抗体IgG则分别为2．83％、3．29％;两者均与氢化可的松及地塞米松无交叉结合反应,与西地兰和地高辛分别存在3．45％vs5．95％、3．20％vs5．20％的交叉反应。结论：IgY比兔抗体ELISA检测内源性哇巴因的特异性及准确性较高。     

In vitro production of feline IgG: quantification by an enzyme-linked immunosorbent assay (ELISA)

We report here on the development of a sensitive and convenient enzyme-linked immunosorbent assay (ELISA) for feline IgG by using commercially available reagents and optimizing their concentrations. The reagents employed include goat anti-cat IgG antibody and alkaline phosphatase-conjugated goat anti-cat IgG antibody. The assay described is sensitive, reproducible, and highly specific for feline IgG. The assay was applied for the measurement of feline IgG synthesized and secreted in vitro by peripheral blood mononuclear cells cultured with or without a polyclonal B-cell activator. The amounts of secreted IgG in the supernatants measured by an ELISA correlated well with the numbers of IgG-secreting cells which were induced upon stimulation with pokeweed mitogen and determined by a reverse hemolytic plaque assay.     

Detection of Bacillus cereus flagellar antigen by enzyme-linked immunosorbent assay (ELISA)

A serological typing scheme of Bacillus cereus has been developed by immunochemical analyses of flagellar antigen using an agglutination method. Enzyme-linked immunosorbent assay (ELISA) for the classification of flagellar serotype of Bacillus cereus had greater sensitivity. 10-500 times, than that of agglutination method. The specificity of flagellar antigen and antibody was determined by immunogold electron microscopy and ELISA inhibition assay. Application of ELISA is useful for the detection of the small amounts and many kinds of antigen-antibody reactions.     

Development of an antibody-based capture enzyme-linked immunosorbent assay for detecting Echinostoma caproni (Trematoda) in experimentally infected rats: kinetics of coproantigen excretion

The present study reports on the development of a coproantigen capture enzyme-linked immunosorbent assay (ELISA) for detecting Echinostoma caproni in experimentally infected rats. The capture ELISA was based on polyclonal rabbit antibodies that recognize excretory-secretory (ES) antigens. The detection limit of pure ES was 3 ng/ml in sample buffer and 60 ng/ml in fecal samples. The test was evaluated using a follow-up of 10 rats experimentally infected with 100 metacercariae of E. caproni, and the results were compared with those of other diagnostic methods such as parasitological examination and antibody titers determined by indirect ELISA. Coproantigens were detected in all the infected rats from the first day postinfection (DPI). The period of maximal coproantigen excretion was between 7 and 21 DPI. The values remained positive until 49-56 DPI, coinciding with the disappearance of the eggs in the stool samples of the infected rats. The kinetics of coproantigen detection were correlated with those of egg output. The present assay provides an alternative tool for the diagnosis of the echinostome infections. The proposed capture ELISA makes possible an earlier diagnosis than that provided by parasitological examination and indirect ELISA and also allows for the differentiation of past and current infections. Our results show that this assay can also be used to monitor the course of echinostome infections.     

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of praziquantel concentration in serum

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of praziquantel in the serum was developed. Since praziquantel has no functional group to conjugate with carrier protein, praziquantel was first converted to a compound with an amino group similar to praziquantel. This compound was then conjugated to bovine serum albumin for use as an immunogen, and to horseradish peroxidase, as enzyme-labeled praziquantel, respectively. The conjugate of praziquantel-bovine serum albumin conjugate was used to raise anti-praziquantel antiserum in mice. The direct competitive ELISA was conducted by simultaneously incubating praziquantel and horseradish peroxidase-labeled praziquantel conjugate with anti-praziquantel antiserum over a second antibody and the enzyme activity of the remaining horseradish peroxidase-labeled praziquantel conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0 to 30 ng ml(-1), and the limit of the detection was 0.3 ng ml(-1). The cross reactivities of anti-praziquantel antibody with compounds related to praziquantel were negligible. Using this ELISA, serum levels of praziquantel were easily determined in male Wistar rats up to 8 h following a single intraperitoneal injection at 2 mg kg(-1) of body weight.     

Anti-galactocerebroside antibodiesin human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA)

The standard ELISA technique was improved for the detection of antigalactocerebroside antibody in biological fluid. Mouse monoclonal antigalactocerebroside antibody was used to demonstrate specificity and sensitivity of the technique. After optimization of the assay, the usefulness of this measurement for the evaluation of patients with multiple sclerosis was assessed. The presence of antigalactocerebroside antibodies in the cerebrospinal fluid of 20 patients with multiple sclerosis, 10 with other neurological diseases and 10 normal individuals was determined. All the CSF samples from normal individuals were negative. In patients with multiple sclerosis 14 of the 20 samples had elevated levels of antigalactocerebroside antibody, whereas with other neurological diseases 5 out of 10 were positive. Antigalactocerebroside levels were lower in samples from patients during an acute relapse than in those from more chronic cases. These results indicate that the presence of anti-galactocerebroside antibody in cerebrospinal fluid is not specific to MS but may reflect previous damage to myelin.Abbreviations and trivial names used ELISA Enzyme-Linked Immunosorbent Assay - CSF cerebrospinal fluid; galacto- or glucocerebroside, ceramide-1-0-beta-galactoside or-glucoside     

Enzyme-linked immunosorbent assay for detection of chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 microg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.     

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

麦长管蚜ELISA检测体系的建立

应用麦长管蚜Macrosiphum avenae(Fabricius)单克隆抗体比较直接、间接和夹心ELISA方法。结果表明,间接ELISA的灵敏度高于直接ELISA,灵敏度分别为0.042μg/mL和0.083μg/mL,而双抗夹心ELISA灵敏度很差,无法使用。建立用于检测捕食性瓢虫肠道内麦长管蚜抗原的间接ELISA检测体系,为进一步应用单克隆抗体评价捕食性瓢虫对麦长管蚜的控制作用奠定可靠的基础。     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Serodiagnosis of M. pneumoniae infections by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA and its sensitivity compared with other serological methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and radioimmunoprecipitation (RIP) are reported. ELISA and MC showed greater sensitivity than CF and MI, while RIP showed serum titer two- to 16-fold higher. ELISA was specific as determined using other human mycoplasma. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution has been proposed. ELISA presented several advantages: sensitivity, rapidity, and low cost and, if adequately standardized, could become a reliable method for the serodiagnosis of M. pneumoniae infection.     

Enhanced chemiluminescent immunoassay for aldosterone

A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%–7.3% in the concentration range 140–1180 pmol/l. The assay showed a significant correlation (r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit.     

Antigen- versus antibody-immobilized ELISA procedures based on a biotinyl-estradiol conjugate

A biotinyl-6 alpha-estradiol derivative (Bio-E2) was synthesized and used as the key component in antigen- and antibody-immobilized ELISA techniques, and the relative merits of the two methods were compared. A precise and reproducible antigen-immobilization was achieved in avidin-coated microtiter plates with Bio-E2. This assay, when completed by the incubation with primary antibody and second antibody-peroxidase conjugate, has a very low detection limit (6 pg/ml estradiol) but required a long incubation time with primary antibody to reach equilibrium. At non-equilibrium conditions, using a high antibody concentration, the assay could be very fast and sensitive. In the antibody-immobilized assay, the Bio-E2 was added to compete with the estradiol present in the calibrator or sample and visualized with a streptavidin-peroxidase conjugate. The detection limit is higher (34 pg/ml), but the specificity was superior and the incubation time to reach equilibrium shorter as compared to the antigen-immobilized assay. Therefore, the antibody-immobilized assay appeared to be ideal for the classical ELISA technique, whereas the antigen-immobilized method seemed to be best suited for automated assay systems using antibody in excess.